EP2398821B1 - Vaccin à virus inactivé contre la dengue, contenant un adjuvant sans aluminium - Google Patents

Vaccin à virus inactivé contre la dengue, contenant un adjuvant sans aluminium Download PDF

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EP2398821B1
EP2398821B1 EP10709443.5A EP10709443A EP2398821B1 EP 2398821 B1 EP2398821 B1 EP 2398821B1 EP 10709443 A EP10709443 A EP 10709443A EP 2398821 B1 EP2398821 B1 EP 2398821B1
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dengue
virus
immunogenic composition
adjuvant
use according
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EP2398821A1 (fr
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Benoit Baras
Dirk Gheysen
Isabelle Solange Lucie Knott
Jean-Paul Prieels
Jean-Francois Toussaint
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GlaxoSmithKline Biologicals SA
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GlaxoSmithKline Biologicals SA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • compositions that elicit an immune response against dengue virus. More specifically, this disclosure concerns inactivated dengue virus vaccines that include an adjuvant.
  • Dengue is an acute viral disease of man which is transmitted by mosquitos. It is endemic in the tropics and subtropics, worldwide, where an estimated 100,000,000 cases occur annually Although relatively rare, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are significant causes of death in children. At present, there is no vaccine to protect against dengue and attempts to prevent disease by controlling the mosquito vector have proven largely ineffective. Thus, there remains a need for a safe and effective vaccine to protect against disease caused by dengue virus. US6254873 discloses an inactivated dengue virus vaccine to immunize and protect humans against dengue fever.
  • the present invention provides an immunogenic composition for use in administration to a human comprising at least one whole killed dengue virus and an aluminum-free adjuvant, wherein the adjuvant is an oil-in-water emulsion comprising squalene, alpha-tocopherol and polyoxyethylene sorbitan monooleate, and wherein the oil-in-water emulsion does not contain any additional immunostimulants.
  • the adjuvant is an oil-in-water emulsion comprising squalene, alpha-tocopherol and polyoxyethylene sorbitan monooleate, and wherein the oil-in-water emulsion does not contain any additional immunostimulants.
  • the present invention is directed to a vaccine that satisfies the need for a safe and effective dengue vaccine.
  • Purified inactivated dengue virus vaccine has a major advantage over live-attenuated dengue virus in that inactivated viruses are not infectious and therefore, can not revert to virulence or cause disease.
  • One potential drawback of a purified inactivated dengue virus vaccine as compared to a live-attenuated is a reduced capacity to induce high titers of dengue specific neutralizing antibodies and the relatively short duration of the protective immune response. These drawback are overcome by the formulation of the purified inactivated dengue antigen with an appropriate adjuvant.
  • the immunogenic compositions disclosed herein include at least one (that is, one or more than one) whole killed dengue virus, in combination with the aluminum-free adjuvant.
  • the whole killed dengue virus can be selected from a Dengue-1 virus, a Dengue-2 virus, a Dengue-3 virus and a Dengue-4 virus.
  • the immunogenic composition can be a monovalent composition including a single whole killed dengue virus from a single strain selected from Dengue-1, Dengue-2, Dengue-3 or Dengue-4, or the composition can be a multivalent (e.g., bivalent, trivalent, tetravalent) composition containing whole killed dengue virus of more than one of these dengue strains.
  • the immunogenic composition includes a whole killed Dengue-2 virus.
  • the immunogenic composition can be a monovalent composition that contains a whole killed Dengue-2 antigen.
  • the immunogenic composition can be a bivalent, trivalent or tetravalent composition that contains a whole killed Dengue-2 virus in combination with one, two or three additional whole killed dengue virus.
  • the composition is a tetravalent composition that includes a whole killed Dengue-1 virus, a whole killed Dengue-2 virus, a whole killed Dengue-3 virus antigen and a whole killed Dengue-4 virus.
  • the one or more whole killed dengue virus are formulated with the adjuvant that is free of aluminum or aluminum salts and wherein the adjuvant is an oil-in-water emulsion comprising squalene, alpha-tocopherol and polyoxyethylene sorbitan monooleate, and wherein the oil-in-water emulsion does not contain any additional immunostimulants.
  • the oil-in-water emulsion also contains an aqueous component, such as a buffered saline solution (e.g., phosphate buffered saline).
  • the oil-in-water emulsion contains squalene and alpha tocopherol in a ratio which is equal or less than 1 (w/w).
  • the immunogenic composition includes an oil-in-water emulsion adjuvant system formulated in a dose comprising: from about 2% to about 10% squalene; from about 2% to about 10% alpha-tocopherol; and from about 0.3% to about 3% polyoxyethylene sorbitan monooleate.
  • the immunogenic composition can include an adjuvant formulated in a dose comprising: from about 10 mg to about 12 mg squalene; from about 10 mg to about 12 mg alpha-tocopherol; and from about 4 mg to about 6 mg polyoxyethylene sorbitan monooleate.
  • the adjuvant includes in a single (whole) dose: 10.68 mg squalene; 11.86 mg tocopherol; 4.85 mg polyoxyethylene sorbitan monooleate.
  • the immunogenic composition is formulated with a fractional dose (that is a dose, which is a fraction of the preceding single dose formulations, such as one half of the preceding quantity of components, 1 ⁇ 4 of the preceding quantity of components, or another fractional dose, e.g., 1/3, 1/6, etc.) of the preceding quantity of components.
  • this disclosure concerns a method for producing a dengue vaccine comprising the following steps: providing at least one purified whole killed dengue virus; and, formulating the at least one purified whole killed dengue virus with an aluminum-free adjuvant, wherein the adjuvant is an oil-in-water emulsion comprising squalenel, alpha-tocopherol and polyoxyethylene sorbitan monooleate and wherein the oil-in-water emulsion does not contain any additional immunostimulants.
  • the immunogenic composition can be formulated with a whole virus produced from either a virulent or attenuated strain, which has been killed.
  • the live (virulent or attenuated) virus can be killed, rendering it incapable of replication, using chemical agents, such as formaldehyde, betapropiolactone (BPL), or hydrogen peroxide, or using ultraviolet irradiation, or by using a combination of two or more inactivation steps (which can be the same or different, e.g., formaldehyde and BPL, formaldehyde and UV irradiation, BPL and UV irradiation, hydrogen peroxide and BPL, hydrogen peroxide and UV irradiation, etc., in any combination).
  • chemical agents such as formaldehyde, betapropiolactone (BPL), or hydrogen peroxide
  • ultraviolet irradiation or by using a combination of two or more inactivation steps (which can be the same or different, e.g., formaldehyde and BPL, formaldehyde and UV irradiation, BPL and UV irradiation, hydrogen peroxide and BPL, hydrogen peroxide and
  • the immunogenic composition may be administered to a child, such as a child under 5 years or age, or under 1 year of age.
  • the immunogenic composition may be administered to a naive subject under 1 year of age.
  • the immunogenic composition may be administered to an adult subject, such as an elderly subject over about 60 or 65 years of age. Such subjects can be previously exposed to dengue virus.
  • the vaccine is administered parentally, e.g., intramuscularly.
  • the immunogenic composition may be used in medicine, e.g., for the prevention, ameleioration or treatment of Dengue virus infection and/or Dengue virus induced disease, such as hemorrhagic fever (DHF) and dengue shock syndrome (DSS).
  • DHF hemorrhagic fever
  • DFS dengue shock syndrome
  • an "immunogenic composition” is a composition of matter suitable for administration to a human or animal subject (e.g., in an experimental setting) that is capable of eliciting a specific immune response, e.g., against a pathogen, such as dengue virus.
  • an immunogenic composition includes one or more antigens (for example, whole purified virus or antigenic subunits, e.g., polypeptides, thereof) or antigenic epitopes.
  • An immunogenic composition can also include one or more additional components capable of eliciting or enhancing an immune response, such as an excipient, carrier, and/or adjuvant.
  • immunogenic compositions are administered to elicit an immune response that protects the subject against symptoms or conditions induced by a pathogen.
  • immunogenic composition will be understood to encompass compositions that are intended for administration to a subject or population of subjects for the purpose of eliciting a protective or palliative immune response against dengue (that is, vaccine compositions or vaccines).
  • purification refers to the process of removing components from a composition, the presence of which is not desired. Purification is a relative term, and does not require that all traces of the undesirable component be removed from the composition. In the context of vaccine production, purification includes such processes as centrifugation, dialization, ion-exchange chromatography, and size-exclusion chromatography, affinity-purification or precipitation. Thus, the term “purified” does not require absolute purity; rather, it is intended as a relative term.
  • a purified virus preparation is one in which the virus is more enriched than it is in its generative environment, for instance within a cell or population of cells in which it is replicated naturally or in an artificial environment.
  • a preparation of substantially pure viruses can be purified such that the desired virus or viral component represents at least 50% of the total protein content of the preparation.
  • a substantially pure virus will represent at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the total protein content of the preparation.
  • an "isolated" biological component such as a virus, nucleic acid molecule, protein or organelle
  • Viruses and viral components e.g., proteins, which have been “isolated” include viruses, and proteins, purified by standard purification methods.
  • the term also embraces viruses and viral components (such as viral proteins) prepared by recombinant expression in a host cell.
  • an “antigen” is a compound, composition, or substance that can stimulate the production of antibodies and/or a T cell response in an animal, including compositions that are injected, absorbed or otherwise introduced into an animal.
  • the term “antigen” includes all related antigenic epitopes.
  • the term “epitope” or “antigenic determinant” refers to a site on an antigen to which B and/or T cells respond.
  • the "dominant antigenic epitopes” or “dominant epitope” are those epitopes to which a functionally significant host immune response, e.g., an antibody response or a T-cell response, is made.
  • the dominant antigenic epitopes are those antigenic moieties that when recognized by the host immune system result in protection from disease caused by the pathogen.
  • T-cell epitope refers to an epitope that when bound to an appropriate MHC molecule is specifically bound by a T cell (via a T cell receptor).
  • B-cell epitope is an epitope that is specifically bound by an antibody (or B cell receptor molecule).
  • a dengue virus is a whole killed virus.
  • adjuvant is an agent that enhances the production of an antigen-specific immune response as compared to administration of the antigen in the absence of the agent.
  • adjuvants include aluminum containing adjuvants that include a suspensions of minerals (or mineral salts, such as aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate) onto which antigen is adsorbed.
  • the adjuvants are aluminum- (alum-)free adjuvants, which are formulated in the absence of any such aluminum salts.
  • Alum-free adjuvants include oil and water emulsions, such as water-in-oil, and oil-in-water (and variants therof, including double emulsions and reversible emulsions), liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll-like Receptor agonists (particularly, TLR2, TLR4, TLR7/8 and TLR9 agonists), and various combinations of such components.
  • oil and water emulsions such as water-in-oil, and oil-in-water (and variants therof, including double emulsions and reversible emulsions)
  • liposaccharides such as water-in-oil, and oil-in-water (and variants therof, including double emulsions and reversible emulsions)
  • liposaccharides such as CpG
  • an “immune response” is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus.
  • An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen specific neutralizing antibodies.
  • An immune response can also be a T cell response, such as a CD4+ response or a CD8+ response. In some cases, the response is specific for a particular antigen (that is, an "antigen-specific response").
  • the antigen-specific response is a "pathogen-specific response.”
  • a "protective immune response” is an immune response that inhibits a detrimental function or activity of a pathogen, reduces infection by a pathogen, or decreases symptoms (including death) that result from infection by the pathogen.
  • a protective immune response can be measured, for example, by the inhibition of viral replication or plaque formation in a plaque reduction assay or ELISA-neutralization assay, or by measuring resistance to pathogen challenge in vivo.
  • a “Th1" biased immune response is characterized by the presence of CD4+ T helper cells that produce IL-2 and IFN- ⁇ , and thus, by the secretion or presence of IL-2 and IFN- ⁇ .
  • a “Th2" biased immune response is characterized by a preponderance of CD4+ helper cells that produce IL-4, IL-5, and IL-13.
  • a "subject” is a living multi-cellular vertebrate organism.
  • the subject can be an experimental subject, such as a non-human animal, e.g., a mouse, a cotton rat, or a non-human primate.
  • the subject can be a human subject.
  • the immunogenic compositions disclosed herein are suitable for preventing, ameliorating and/or treating disease caused by infection with dengue virus.
  • the immunogenic compositions disclosed herein include one or more purified whole killed dengue virus.
  • the immunogenic compositions can include a single strain of dengue virus (i.e., a monovalent composition), or they can contain more than one strain of dengue virus (i.e., a multivalent composition).
  • a multivalent composition contains strains selected from different serotypes.
  • the immunogenic composition is a tetravalent composition that includes strains selected from each of the four serotypes of dengue virus.
  • the viruses used as antigens can be selected from essentially any strain (or strains) of dengue virus.
  • a virus strain can be selected for each serotype, which is chosen based on its conformity to a defined (e.g., consensus) sequence for the serotype, such as a DEN-1 consensus sequence, a DEN-2 consensus sequence, a DEN-3 consensus sequence, or a DEN-4 consensus sequence.
  • a virus strain can be selected to correlate with a strain prevalent in the area or population in which the vaccine is intended to be administered.
  • Another option is to select strains for each serotype as a matter of convenience based on availability or prior experience. For example, exemplary strains are described in US Patent No.
  • virulent or attenuated strains can be used.
  • virulent strains propagate to higher titer in host cells, facilitating production at commercial scale.
  • virulent strains require special care in handling to prevent infection of personnel involved in manufacturing.
  • Attenuated strains e.g., developed by adaptation to production in cultured cells and selection for reduced virulence and/or reduced replication in the mosquito vectors of dengue, require fewer handling precautions but can be difficult to produce.
  • Exemplary attenuated strains suitable for use in the context of an immunogenic composition containing an inactivated dengue virus and an aluminum-free adjuvant are described in WO 00/57907 and US Patent No.
  • strain(s) selected are typically chosen from among the numerous strains available to replicate in cells that are suitable for production of materials intended for human use (e.g., cells that are certified free of pathogens).
  • strains can be screened to identify those viruses that grow to the highest titers, for example from a titer of at least about 5x10 6 pfu/ml, preferably at least 1x10 7 pfu/ml or more in the cell line(s) of choice; (ii) selecting those strains of dengue virus which grow to the highest titers in the cell line(s) of choice; and (iii) further adapting those selected strains for enhanced growth by additional passage from one to several times in the cell line(s) of choice.
  • the selected viruses (for example, chosen from the four serotypes of dengue viruses) can be further adapted to grow to high titers by additional cell culture passages or by genetic manipulation to make high-titered master and production seed lots.
  • Suitable cell lines for propagating dengue virus include mammalian cells, such as Vero cells, AGMK cells, BHK-21 cells, COS-1 or COS-7 cells, MDCK cells, CV-1 cells, LLC-MK2 cells, primary cell lines such as fetal Rhesus lung (FRhL-2) cells, BSC-1 cells, and MRC-5 cells, or human diploid fibroblasts, as well as avian cells, chicken or duck embryo derived cell lines, e.g., AGE1 cells, and primary, chicken embryo fibroblasts, and mosquito cell lines, such as C6/36.
  • the chosen cell(s) are adapted to grow in the absence of serum or serum-derived proteins, and can maintain dengue virus replication at high titers under serum-free (and/or protein-free) growth conditions.
  • the selected dengue virus strain is used to infect the host cell (for example, selected from among the suitable cell types listed above).
  • the cultures are fed with medium capable of supporting growth of the cells.
  • the medium does not contain serum or serum-derived proteins or other animal-derived proteins, or serum-free maxima can be used to replace serum-containing media during production.
  • serum-free medium Numerous formulations of serum-free medium are available commercially. A detailed description of methods for producing virus in cells maintained under serum-free conditions can be found, for example, in published US Patent Application No. 20060183224 , which is incorporated herein by reference.
  • the host cells are maintained in culture for several days until the desired virus titer is achieved.
  • the cells are maintained in a continuous perfusion system from which virus can be intermittently or continuously obtained over the course of several days or more.
  • a virus titer of at least about 10 6 to 10 7 PFU/ml by 3-7 days post infection, is desirable.
  • the titer remains high for several days, and virus can be recovered at multiple timepoints to maximize yield.
  • virus can be harvested from these cultures daily, from about 3 to about 13 days post infection by collecting the supernates and refeeding the cells.
  • the supernates can be pooled prior to additional processing.
  • virus can be grown to a higher titer, but over a shorter period of time. In such a case, the virus can be harvested at peak titer as determined empirically.
  • Vero cells Vero cells can be amplified in VPSFM medium with Ficin up to passage 141.
  • One additional passage can be performed in the presence of porcine trypsin.
  • Cells can be seeded at approximately 0.7x10 6 cells/ml in Bioreactor (4L) and cultivated on microcarriers (cytodex-1) for 5 days in VPSFM+ Pluronic 0.1% under perfusion conditions (e.g., D0->: 0 Volume/day; D1->D2: 1 v/d; then 1.5 v/d for the 3 next days).
  • Cells at approximately 3x10 6 cells/ml are then infected (at 35°C) with dengue virus, e.g., at a MOI 0.01 in DMEM+Glutamine 4mM++FBS 2% for 2h with a reduced medium volume (2L) at 37°C.
  • the medium volume in the Bioreactor is then adjusted to 4L. After 3 days, a large part of the medium (around 3L) is removed and replaced by DMEM medium+ Glutamin 4mM. At 7 days post-infection, virus can be harvested.
  • the virus is harvested by common methods known in the art including slow-speed centrifugation (for example at 1500xg for 10 min), or by filtration through a filter of pore size of 0.45 ⁇ m.
  • Methods for concentrating said virus(es) are within the scope of a person with ordinary skill in the art and include, for example, ultrafiltration (e.g., with a membrane of no greater than 300 kDa pore size), or precipitation with polyethelene glycol (PEG) 8000.
  • Methods for purifying viruses include continuous or multi-step sucrose gradients, purification by column chromatography using size exclusion, ion exchange, adsorption, or affinity columns, or purification by partitioning in polymer two-phase or multi-phase systems, and any combination thereof.
  • Methods for assaying for virus positive fractions include plaque assay, hemagglutination (HA) assay, and/or antigen assays such as immunoassays.
  • dengue virus can be concentrated from culture supernate by polyethylene glycol (PEG) precipitation.
  • Supernate fluids from infected cells are clarified by centrifugation (1,500xg) for 10 min.
  • the clarified supernate is adjusted to 6% PEG 8000 (Sigma) and 0.5 M NaCl and held at 4 °C. with gentle stirring for 45 min.
  • the precipitate is collected by centrifugation at 5000xg for 50 min and resuspended in STE buffer (0.1 M NaCl, 10 mM tris, 1 mM EDTA, pH 7.6).
  • the virus suspension can then be clarified by centrifugation (12,000xg for 10 min at 4 °C).
  • the pellet is discarded, and the supernate is then centrifuged (170,000xg, 80 min, 4 °C) to pellet virus.
  • This virus pellet is resuspended in STE buffer at the desired concentration.
  • dengue virus can be concentrated from cell supernates by tangential flow ultrafiltration.
  • Supemate fluids from infected cells is clarified by centrifugation at low speed as described above, then filtered through a 0.45 ⁇ m CN filter (Nalgene).
  • the filtered supernate is concentrated by tangential flow ultrafiltration using a low-protein-binding 100 kDa-cutoff membrane (e.g., omega 100K screen channel, Filtron, Inc.). Concentration is carried out at 4 °C. using a flow rate of 400 ml per min, a filtration rate of approximately 100 ml per min and a pressure of 20-30 psi.
  • Dengue virus can be purified on sucrose gradients essentially as described previously ( Srivastava et al. Arch. Virol. 96: 97-107, 1987 ) with minor modifications.
  • sucrose gradients can be made in 1 "x3.5" (40 ml) ultracentifuge tubes (Ultra-clear.TM., Beckman, Inc.) by stepwise addition of the following w/w sucrose solutions in phosphate buffered saline, pH 7.4 (PBS, without Ca and Mg, Whittaker MA Bioproducts): 2 ml 60%, 2 ml 55%, 2 ml 50%, 2 ml 45%, 2 ml 40%, 2 ml 35%, 2 ml 30% and 1 ml 15%.
  • a smooth gradient is formed by allowing the tubes to stand for 2-4 hrs at room temperature. Up to 25 ml of concentrated virus is applied to each tube.
  • Ultracentrifugation is then carried out in (e.g., in a SW 28 rotor (Beckman) at 17,000 rpm for 18 hrs at 4 °C). Following centrifugation, 1 to 2 ml fractions can be collected from the bottom of the tubes. Fractions can be assayed for total protein, virus HA, and virus antigen as desired. Positive gradient fractions are typically pooled, and diluted to 10% or less sucrose with Medium 199 (M199, Gibco-BRL) or PBS. Optionally, prior to inactivation, virus pools can be filtered through a 0.22 ⁇ m low-protein-binding filter (GV type, Millipore).
  • GV type 0.22 ⁇ m low-protein-binding filter
  • the recovered viruses are inactivated by a means selected to preserve their antigenicity and immunogenicity while destroying their infectivity.
  • an agent such as formalin or beta-propriolactone is added to the virus, and the mixture is incubated with the inactivating agent until inactivated.
  • formalin 37% formaldehyde
  • the solution is then sterilized by passage through a 0.22 ⁇ m CN filter (Nalgene). This formalin solution is added to purified virus (1:50) for a final formalin concentration of 0.05%.
  • Inactivation is carried out at between 15 and 25 °C, for 48 hrs up to 14 days (typically, 10 days or less).
  • virus is filtered through a 0.22 um GV type filter and transferred to a fresh container.
  • free formalin in the bulk culture is neutralized, e.g., by addition of an equimolar amount of sterile 10% w/v sodium bisulfite.
  • inactivation can be achieved by irradiating the virus with a radioactive source until the virus is inactivated.
  • a radioactive source is cobalt-60, at doses sufficient to inactivate the infectivity of the viruses while preserving the antigenicity essentially intact. Examples of useful doses are those which fall within the range from 5.5 to 7.0 Mrads.
  • virus aliquots of 50 ⁇ l in 1.5 ml sterile polypropylene tubes are frozen and placed on dry ice in the gamma cell of a 60 Co source, for a period sufficient to deliver the desired irradiation.
  • ultraviolet irradiation can be employed to inactivate dengue virus.
  • Suitable devices for ultraviolet irradiation of virus in a commercial setting are well known in the art, and devices are commercially available, e.g., from Bayer, which expose the supernate (or other fluid containing the virus) to a UV-C light source at approximately 254 nm, for a time sufficient to inactivate virus while preserving immunogenicity.
  • the dengue virus can be inactivated using hydrogen peroxide as described, e.g., in published US Patent Application No. 20070031451 , which is incorporated herein by reference.
  • two or more activation steps can be employed.
  • steps can be the same or different.
  • a combination of any suitable inactivation process, such as any of the preceding inactivation processes, can be employed during the purification and inactivation of dengue virus for formulation into an immunogenic composition for administration to a human subject.
  • virus can be monitored prior to inactivation in a plaque titration assay.
  • Virus is amplified on the same or a different host cell as used for production, and used to infect a suitable cell line, such as LLC-MK2 cells or Vero cell monolayers, and the number of viral plaques can be assessed to determine infectivity of the recovered virus (see, e.g., Sukhavachanaet al. WHO Bull. 35: 65-6, 1966 ).
  • Another method for assessing quantity and quality of recovered antigen is by virus hemagglutination (HA) and hemagglutination-inhibition (HI) assays.
  • Virus HA and HI assays can be performed as previously described ( Clarke & Casals, Am. J. Trop. Med. Hyg. 7: 561-73, 1958 ). Total protein is determined essentially as described by Bradford (Anal. Biochem. 72: 248, 1976 ), using a commercially-available kit (BioRad, Hercules, Calif.) and bovine serum albumin (BSA) or gamma globulin as a standard.
  • BSA bovine serum albumin
  • antigen can be detected and/or quantitated after inactivation, e.g., in an antigen spot blot assay.
  • virus samples are diluted out serially, typically by two-fold dilution, and spotted onto nitrocellulose paper. The papers are air-dried, blocked with 5% casein in PBS, and incubated with a specific antibody or antiserum followed by enzyme-linked secondary antibody. Virus can also be detected by western blotting.
  • antigen preparations are solubilized in SDS-PAGE sample buffer containing 1% SDS, 66 mM Tris-HCl, pH 6.8, 1% glycerol and 0.7% bromphenol blue at 22 C.
  • the whole killed dengue virus(es) is mixed with the aluminum-free adjuvant to produce an immunogenic composition suitable for immunizing human subjects in order to elicit high titers of virus neutralizing antibodies and protect the immunized human from disease caused by dengue virus.
  • the whole killed dengue virus(es) are formulated in a pharmaceutically acceptable carrier or excipient.
  • the carrier or excipient can favorably include a buffer.
  • the carrier or excipient also contains at least one component that stabilizes solubility and/or stability.
  • solubilizing/stabilizing agents include detergents, for example, laurel sarcosine and/or polyoxyethylene sorbitan monooleate.
  • Alternative solubilizing/stabilizing agents include arginine, and glass forming polyols (such as sucrose, trehalose and the like).
  • Numerous pharmaceutically acceptable carriers and/or pharmaceutically acceptable excipients are known in the art and are described, e.g., in Remihgtoh's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 5th Edition (975 ).
  • suitable excipients and carriers can be selected by those of skill in the art to produce a formulation suitable for delivery to a subject by a selected route of administration.
  • Suitable excipients include, without limitation: glycerol, Polyethylene glycol (PEG), Sorbitol, Trehalose, N-lauroylsarcosine sodium salt, L -proline, Non detergent sulfobetaine, Guanidine hydrochloride, Urea, Trimethylamine oxide, KCl, Ca 2+ , Mg 2+ , Mn 2+ , Zn 2+ and other divalent cation related salts, Dithiothreitol, Dithioerytrol, and ß-mercaptoethanol.
  • PEG Polyethylene glycol
  • Sorbitol Sorbitol
  • Trehalose N-lauroylsarcosine sodium salt
  • L -proline Non detergent sulfobetaine
  • Urea Trimethylamine oxide
  • KCl Ca 2+ , Mg 2+ , Mn 2+ , Zn 2+ and other divalent cation related salts
  • Dithiothreitol Dithioery
  • excipients can be detergents (including: polyoxyethylene sorbitan monooleate, Triton X-00, NP-40, Empigen BB, Octylglucoside, Lauroyl maltoside, Zwittergent 3-08, Zwittergent 3-0, Zwittergent 3-2, Zwittergent 3-4, Zwittergent 3-6, CHAPS, Sodium deoxycholate, Sodium dodecyl sulphate, Cetyltrimethylammonium bromide).
  • detergents including: polyoxyethylene sorbitan monooleate, Triton X-00, NP-40, Empigen BB, Octylglucoside, Lauroyl maltoside, Zwittergent 3-08, Zwittergent 3-0, Zwittergent 3-2, Zwittergent 3-4, Zwittergent 3-6, CHAPS, Sodium deoxycholate, Sodium dodecyl sulphate, Cetyltrimethylammonium bromide).
  • the immunogenic compositions disclosed herein also include an adjuvant.
  • the adjuvant is an aluminum-free adjuvant, wherein the adjuvant is an oil-in-water emulsion comprising squalene, alpha-tocopherol and polyoxyethylene sorbitan monooleate, and wherein the oil-in-water emulsion does not contain any additional immunostimulants.
  • the aqueous carrier can be, for example, phosphate buffered saline.
  • An immunogenic composition typically contains an immunoprotective quantity (or a fractional dose thereof) of the antigen and can be prepared by conventional techniques.
  • Preparation of immunogenic compositions including those for administration to human subjects, is generally described in Pharmaceutical Biotechnology, Vol.61 Vaccine Design-the subunit and adjuvant approach, edited by Powell and Newman, Plenurn Press, 1995 . New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978 .
  • Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877 .
  • Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757 .
  • the amount of antigen in each dose of the immunogenic composition is selected as an amount which induces an immunoprotective response without significant, adverse side effects in the typical subject.
  • Immunoprotective in this context does not necessarily mean completely protective against infection; it means protection against symptoms or disease, especially severe disease associated with the virus.
  • the amount of antigen can vary depending upon which specific immunogen is employed.
  • each human dose will comprise 0.05-100 ⁇ g of inactivated virus, such as from about 0.1 ⁇ g (e.g., 0.1, 0.2, 0.3, 0.4, or 0.5 ⁇ g) to about 50 ⁇ g, for example, from about 0.5 ⁇ g to about 30 ⁇ g, such as about 1 ⁇ g, about 2 ⁇ g, about 3 ⁇ g, about 4 ⁇ g, about 5 ⁇ g, about 10 ⁇ g, about 15 ⁇ g, about 20 ⁇ g, or about 25 ⁇ g, of each strain of inactivated dengue virus.
  • the amount utilized in an immunogenic composition is selected based on the subject population (e.g., infant). An optimal amount for a particular composition can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects can receive a boost after a suitable interval (e.g., in about 4 weeks).
  • the concentration in the final formulation is calculated to be safe and effective in the target population.
  • immunogenic compositions for eliciting an immune response against dengue virus in humans are favorably administered to infants (e.g., infants between birth and 1 year, such as between 0 and 6 months, at the age of initial dose).
  • Immunogenic compositions for eliciting an immune response against dengue are also favorably administered to adult humans (e.g., alone or in a combination with antigens of other pathogens for example in the context of a "traveler's" vaccine).
  • the choice of adjuvant can be different in these different applications, and the optimal adjuvant and concentration for each situation can be determined empirically by those of skill in the art.
  • vaccines are prepared as injectables, either as liquid solutions or suspensions; solid form suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the composition can be administered by a variety of different routes, most commonly, the immunogenic compositions are delivered by an intramuscular, subcutaneous or intradermal route of administration. Generally, the vaccine may be administered subcutaneously, intradermally, or intramuscularly in a dose effective for the production of neutralizing antibody and protection.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
  • the quantity to be administered which is generally in the range of 0.05-100 ⁇ g of each strain of inactivated virus per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of the vaccine to be administered may depend on the judgment of the practitioner and may be peculiar to each subject.
  • the vaccine may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months or years.
  • the dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
  • suitable immunization schedules include: a first dose, followed by a second dose between 7 days and 6 months, and an optional third dose between 1 month and two years post initial immunization, or other schedules sufficient to elicit titers of virus-neutralizing antibodies expected to confer protective immunity, for example selected to correspond to an established pediatric vaccine schedule.
  • the generation of protective immunity against dengue with an inactivated virus vaccine may reasonably be expected after a primary course of immunization consisting of 1 to 3 inoculations. These could be supplemented by boosters at intervals (e.g., every two years) designed to maintain a satisfactory level of protective immunity.
  • Example I Immunogenicity of exemplary immunogenic compositions that contain a Dengue purified inactivated virus (1 ⁇ g nd 10 ⁇ g) and an aluminum-free adjuvant.
  • mice Groups of 15 naive adult female C57B1/6 mice were vaccinated intramuscularly with two doses of an exemplary vaccine candidate containing purified inactivated virus (PIV) of a Dengue-2 (Den-2) strain.
  • the vaccine was administered in a total volume of 50 ⁇ l.
  • mice were immunized with formulations containing Dengue PIV alone or formulations containing Dengue PIV vaccine adjuvanted with Alum (alum hydroxide), 3-Deacylated monophoshoryl lipid A (3D-MPL) adsorbed onto aluminum hydroxide (AS04D), an oil-in-water emulsion (AS03A), and 3D-MPL and QS21 in a liposomal formulation (AS01B) (see groups in Table 1 below).
  • AS03A and AS01B are two non-limiting examples of aluminum (alum)-free adjuvants.
  • the Dengue PIV antigen used for formulation was a purified bulk obtained essentially as described herein from a culture of a wild-type Den-2 virus, inactivated with 0.185% of formalin for 7 days at 22°C.
  • PIV Den-2 (Conc Total prot/dose) Other treatment 1 PIV Den-2 Plain (non-adjuvanted) 10 ⁇ g Days 0 and 21 2 PIV Den-2 + AS22A (Al(OH)3) 10 ⁇ g Days 0 and 21 3 PIV Den-2 + AS04D 10 ⁇ g Days 0 and 21 4 PIV Den-2 + AS03A 10 ⁇ g Days 0 and 21 5 PIV Den-2 + AS01B 10 ⁇ g Days 0 and 21 6 PIV Den-2 + Plain (non-adjuvanted) 1 ⁇ g Days 0 and 21 7 PIV Den-2 + AS22A (Al(OH)3) 1 ⁇ g Days 0 and 21 8 PIV Den-2 + AS04D 1 ⁇ g Days 0 and 21 9 PIV Den-2 + AS03A 1 ⁇ g Days 0 and 21 10 PIV Den-2 + AS01B 1 ⁇ g Days 0 and 21 11 PBS Days 0 and 21
  • the humoral immune response was measured 21 days after the first immunization (set as Day 0) and the second immunization (Day 21), that is, on days 21 and 42, post immunization. Serum samples were tested using an anti-Dengue neutralization assay. In brief, to measure mouse serum neutralizing titers against Dengue virus, serial dilutions of filtered and heat inactivated serum were incubated with a fixed amount of monospecific Dengue virus (Dengue-2). The mixture of serum and virus was then added to a monolayer of Vero cells (from a WHO cell bank) and incubated for 4 days.
  • Viral infection inhibition by serum samples was measured using an ELISA that detects cellassociated viral antigens on Vero cells adhered to a 96-well microplate.
  • the resulting optical density readings were automatically processed into an Excel spreadsheet that uses a log midpoint linear regression program model to derive a virus percent reduction of infection (referred to as microneutralization 50 percent reduction, or MN50).
  • MN50 virus neutralization titer
  • the virus neutralization titer (MN50) is defined as the reciprocal of the serum dilution giving 50% reduction in the absorbance readout of the assay when compared to the virus dose control without serum (TV).
  • Neutralizing antibody titers were determined on pooled sera for each group at day 21, and on individual sera at day 42. Results are illustrated in FIG. 1 . The same immunological profile was observed for both doses tested (1 and 10 ⁇ g total protein). The neutralizing antibody titers were enhanced after two administrations of 1 ⁇ g or 10 ⁇ g of Dengue PIV vaccine as compared to the response after a single dose of the composition. For both doses of Dengue PIV vaccine tested, significantly lower neutralizing antibody responses were observed with the non-adjuvanted vaccine compared to the adjuvanted Dengue PIV vaccine (p ⁇ 0.00001).
  • compositions adjuvanted with either alum-free adjuvant induced significantly higher neutralizing antibody responses (AS03A (p ⁇ 0.0173) or AS01B (p ⁇ 0.0001)) than did the compositions with AS04D or AS22A.
  • Dengue PIV vaccine adjuvanted with AS03A and AS01B induced similar levels of neutralizing antibody titers (p>0.05).
  • Example II Immunogenicity of exemplary immunogenic compositions that contain a Dengue purified inactivated virus (1 ⁇ g nd 10 ⁇ g) and an aluminum-free adjuvant at different dilutions of adjuvant.
  • mice Groups of 15 adult female C57B1/6 mice were administered two doses of the exemplary Dengue PIV vaccine intramuscularly in a total volume of 50 ⁇ l. Mice were immunized with formulations containing either Dengue PIV alone or formulations containing Dengue PIV antigen adjuvanted with Alum (AS22A, alum hydroxide), or dilution of different adjuvant systems, e.g., such as 1 ⁇ 2 the standard dose of AS04D (AS04D/2), AS03B (oil-in-water emulsion-based Adjuvant System containing 5.93 mg tocopherol), and AS01E (half dose of AS01B) (detailed in Table 2).
  • AS22A Dengue PIV antigen adjuvanted with Alum
  • AS03B oil-in-water emulsion-based Adjuvant System containing 5.93 mg tocopherol
  • AS01E half dose of AS01B
  • the PIV used for formulation was a purified bulk obtained from a culture of a wild-type Den-2 virus, inactivated with 0.185% of Formalin for 7 days at 22°C.
  • Table 2 Gr Antigen / Formulation PIV Den-2 Conc Total prot/mouse Other treatment 1 PIV Den-2 Plain (non-adjuvanted) 1 ⁇ g Days 0 and 21 2 PIV Den-2 + AS22A (Al(OH)3) 1 ⁇ g Days 0 and 21 5 PIV Den-2 + AS04D/2 10 ⁇ g Days 0 and 21 6 PIV Den-2 + AS03B 10 ⁇ g Days 0 and 21 7 PIV Den-2 + AS01E 10 ⁇ g Days 0 and 21 8 PIV Den-2 + AS04D/2 1 ⁇ g Days 0 and 21 9 PIV Den-2 + AS03B 1 ⁇ g Days 0 and 21 10 PIV Den-2 + AS01E 1 ⁇ g Days 0 and 21 11 PBS Days 0 and 21
  • the humoral immune response to immunization was measured 21 days after the first and second immunizations (Days 21 and 42, respectively) on 15 mice/group. Serum samples were tested by the neutralization assay described above.
  • significantly lower neutralizing antibody responses were observed with the non-adjuvanted vaccine compared to the adjuvanted Dengue PIV formulations (p ⁇ 0.00001).
  • Dengue PIV vaccine adjuvanted with AS04D/2 induced similar neutralizing antibody response compared to the response induced by the Dengue PIV vaccine adjuvanted with alum alone (p>0.05).
  • Dengue PIV vaccine adjuvanted with AS03B or AS01E induced significantly higher neutralizing antibody titers compared to vaccine adjuvanted with AS04D (p ⁇ 0.029 for AS03B and p ⁇ 0.00001 for AS01E) or alum (p ⁇ 0.0035 for AS03B and p ⁇ 0.00001 for AS01E).
  • Example III Immunogenicity of exemplary immunogenic compositions that contain a Dengue purified inactivated virus ((2 ⁇ g and 200 ng) and an aluminum-free adjuvant at different dilutions of adjuvant.
  • mice Groups of 25 adult female C57B1/6 mice were immunized intramuscularly with two doses of an exemplary Dengue PIV vaccine in a total volume of 50 ⁇ l. Mice were immunized with formulations containing Dengue PIV antigen alone or formulations containing Dengue PIV antigen adjuvanted with Alum (AS22A, alum hydroxide) or dilution of different adjuvant, e.g., AS04D (AS04D/2), AS03B (oil-in-water emulsion-based Adjuvant System containing 5.93 mg tocopherol), and AS01E (half dose of AS01B) (the groups are detailed in Table 3).
  • AS22A alum hydroxide
  • AS03B oil-in-water emulsion-based Adjuvant System containing 5.93 mg tocopherol
  • AS01E half dose of AS01B
  • the PIV antigen used for formulation was a purified bulk obtained from a culture of a wild-type Den-2 virus, inactivated with 0.185% of formalin for 7 days at 22°C.
  • Table 3 Gr Antigen / Formulation PIV Den-2 Conc Total prot/mouse Other treatment 1 PIV Den-2 Plain (non-adjuvanted) 200 ng Days 0 and 14 2 PIV Den-2 + AS22A (Al(OH)3) 200ng Days 0 and 14 3 PIV Den-2 + AS04D/2 2 ⁇ g Days 0 and 14 4 PIV Den-2 + AS03B 2 ⁇ g Days 0 and 14 5 PIV Den-2 + AS01E 2 ⁇ g Days 0 and 14 6 PIV Den-2 + AS04D/2 200 ng Days 0 and 14 7 PIV Den-2 + AS03B 200 ng Days 0 and 14 8 PIV Den-2 + AS01E 200 ng Days 0 and 14 9 PBS Days 0 and 14
  • the humoral immune response to administration of the immunogenic composition was measured 14 days after the first and second immunizations (Days 14 and 28, respectively) on 15 mice/group. Serum samples were evaluated by the neutralization assay described above.
  • mice from each group were sacrificed at 7 days post immunization in order to evaluate the cellular immune response by ICS using 5 pools of 2 spleens each.
  • CMI was only evaluated for the groups of mice immunized with 2 ⁇ g of Dengue PIV vaccine adjuvanted with AS04D/2, AS03B, AS01E and in mice immunized with 200 ng of the non-adjuvanted vaccine or the vaccine adjuvanted with AS22A (groups1 to 5) and mice receiving PBS (group 9). Sera of 15 other mice were collected 14 days after the first immunization (day 14) and the second immunization (day 28).
  • Neutralizing antibody titers were determined on pooled sera per group at day 14, and on individual sera per group at day 28. Results are provided in FIG 3 . Higher neutralizing antibody titers were observed after two administrations of either 2 ⁇ g or 200 ng of Dengue PIV vaccine as compared to the titers after a single administration. Significantly higher neutralizing antibody responses were observed with the adjuvanted Dengue PIV formulations as compared to the non-adjuvanted formulation (p ⁇ 0.0089). At the dose of 200 ng of Dengue PIV, the formulations adjuvanted with AS03B and AS01E elicited a substantially higher neutralizing antibody response as compared to the response elicited by the same antigen formulated with AS22A or AS04D/2.
  • Dengue PIV adjuvanted with AS22A and AS04D/2 induced comparable neutralizing antibody response at the dose of 200 ng total protein (p>0.05).
  • the formulation with AS04D/2 induced similar neutralizing antibody response compared to the vaccine adjuvanted with AS03B (p>0.05).
  • Dengue PIV adjuvanted with AS01E induced significantly higher neutralizing antibody titers compared to all other groups (p ⁇ 0.0028).
  • the cellular immune response was also evaluated following two administrations of Dengue PIV in combination with the aforementioned dilutions of adjuvant.
  • CD4+ T cell cytokine responses were evaluated 7 days after the second immunization (Day 21) by ICS ( FIG. 4 ).
  • mice immunized with Dengue PIV formulated with Alum-containing adjuvants (AS22A, AS04D or AS04D/2)
  • mice immunized with a vaccine formulated with alum-free adjuvant systems e.g., AS01B or AS01E, which do not contain alum
  • a vaccine formulated with alum-free adjuvant systems e.g., AS01B or AS01E, which do not contain alum

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Claims (15)

  1. Composition immunogène destinée à être administrée à un sujet humain comprenant au moins un virus entier de la dengue tué et un adjuvant sans aluminium, dans laquelle l'adjuvant est une émulsion huile dans l'eau comprenant du squalène, de l'alpha-tocophérol et du monooléate de polyoxyéthylène-sorbitanne, et dans laquelle l'émulsion huile dans l'eau ne contient aucun immunostimulant supplémentaire.
  2. Composition immunogène pour son utilisation selon la revendication 1, dans laquelle le au moins un virus entier de la dengue tué est choisi dans le groupe constitué par un virus de la dengue 1, un virus de la dengue 2, un virus de la dengue 3 et un virus de la dengue 4.
  3. Composition immunogène pour son utilisation selon la revendication 1 ou 2, dans laquelle le au moins un virus entier de la dengue tué est un virus de la dengue 2.
  4. Composition immunogène pour son utilisation selon l'une quelconque des revendications 1 à 3, comprenant du squalène et de l'alpha-tocophérol dans un rapport qui est égal ou inférieur à 1.
  5. Composition immunogène pour son utilisation selon l'une quelconque des revendications 1 à 4, dans laquelle l'adjuvant est formulé dans une dose comprenant :
    d'environ 2% à environ 10 % de squalène,
    d'environ 2% à environ 10 % d'alpha-tocophérol,
    d'environ 0,3% à environ 3 % de monooléate de polyoxyéthylène-sorbitanne.
  6. Composition immunogène pour son utilisation selon la revendication 5, dans laquelle l'adjuvant est formulé dans une dose comprenant :
    d'environ 10 mg à environ 12 mg de squalène,
    d'environ 10 mg à environ 12 mg d'alpha-tocophérol,
    d'environ 4 mg à environ 6 mg de monooléate de polyoxyéthylène-sorbitanne.
  7. Composition immunogène pour son utilisation selon la revendication 5 ou 6, dans laquelle l'adjuvant est formulé dans une dose comprenant :
    10,68 mg de squalène,
    11,86 mg de tocophérol,
    4,85 mg de monooléate de polyoxyéthylène-sorbitanne.
  8. Composition immunogène pour son utilisation selon la revendication 7, dans laquelle l'adjuvant est formulé dans une dose fractionnée.
  9. Composition immunogène pour son utilisation selon l'une quelconque des revendications 1 à 8, dans laquelle la composition immunogène comprend un virus de la dengue 1, un virus de la dengue 2, un virus de la dengue 3 et un virus de la dengue 4.
  10. Composition immunogène pour son utilisation selon l'une quelconque des revendications 1 à 9, où ladite composition immunogène est une composition vaccinale contre la dengue.
  11. Composition immunogène pour son utilisation selon l'une quelconque des revendications 1 à 10 destinée à être utilisée pour prévenir, améliorer ou traiter une maladie provoquée par le virus de la dengue chez un sujet.
  12. Composition immunogène pour son utilisation selon la revendication 11, où ledit sujet est âgé de moins de cinq ans.
  13. Composition immunogène pour son utilisation selon la revendication 11, où ledit sujet est âgé de moins d'un an.
  14. Composition immunogène pour son utilisation selon l'une quelconque des revendications 11, 12 ou 13, où ladite maladie provoquée par le virus de la dengue est une fièvre hémorragique (DHF) ou la dengue avec syndrome de choc (DSS).
  15. Méthode de production d'un vaccin contre la dengue destiné à être administré à un sujet humain comprenant :
    La provision d'au moins un virus entier de la dengue tué et purifié ; et
    la formulation du au moins un virus entier de la dengue tué et purifié avec un adjuvant sans aluminium, dans laquelle l'adjuvant est une émulsion huile dans l'eau comprenant du squalène, de l'alpha-tocophérol et du monooléate de polyoxyéthylène sorbitanne, et dans laquelle l'émulsion huile dans l'eau ne contient aucun immunostimulant supplémentaire.
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TW201517915A (zh) 2015-05-16
TW201043245A (en) 2010-12-16
EP2398821A1 (fr) 2011-12-28
KR20110132379A (ko) 2011-12-07
PE20151588A1 (es) 2015-11-18
UY32457A (es) 2011-01-31
JP2012517979A (ja) 2012-08-09
CO6551750A2 (es) 2012-10-31
CN102395600B (zh) 2015-03-25
JP2015221803A (ja) 2015-12-10
US20110318407A1 (en) 2011-12-29
KR101738704B1 (ko) 2017-06-08
DOP2011000268A (es) 2012-07-31
ES2649020T3 (es) 2018-01-09
SG2014014385A (en) 2014-04-28
JP6104988B2 (ja) 2017-03-29
CN102395600A (zh) 2012-03-28
CA2752809A1 (fr) 2010-08-26
CL2011001993A1 (es) 2012-04-09
AR075437A1 (es) 2011-03-30
MX338898B (es) 2016-05-03
AU2010215595A1 (en) 2011-08-25
MX2011008649A (es) 2011-09-06
PH12015501637A1 (en) 2019-12-11
IL214385A (en) 2016-11-30
PE20110992A1 (es) 2012-02-12
NZ700477A (en) 2016-04-29
JP6058266B2 (ja) 2017-01-11
WO2010094663A1 (fr) 2010-08-26
ZA201105656B (en) 2012-04-25
TWI528972B (zh) 2016-04-11
IL214385A0 (en) 2011-09-27
BRPI1011224A2 (pt) 2016-03-15
SG173194A1 (en) 2011-09-29
US20160120972A1 (en) 2016-05-05

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